Abstract Chondrodysplasias comprise a class of heritable defects that occur in 1/2500 births and disrupt skeletal growth, resulting in dwarfism or asymmetric limb length. Therapeutic options for chondrodysplasias are limited in part because the underlying mechanisms that regulate cartilage architecture are poorly understood. Anisotropic processes that increase cell volume and deposit cartilage matrix are largely responsible for the growth of long bones. The inherent anisotropies in growth plate cartilage are mirrored by the arrangement of chondrocytes in the developing bone. Specifically, the architecture of cartilage arises from disordered resting progenitor cells that enter a transit amplifying phase in which clonal expansion generates columns of discoid cells that resemble stacks of coins that are aligned with the longitudinal axis of the bone. Column formation occurs in a process involving planar cell division followed by rearrangement of daughter cells. Interestingly, genetic studies in model organisms have shown many chondrodysplasia phenotypes are associated with defects in chondrocyte column formation. These studies also revealed that defects in cell signaling and cell polarity pathways, cell adhesion, and extracellular matrix structure each disrupt column formation and produce chondrodysplasia. However, a major gap in knowledge exists regarding how these distinct molecular functions are integrated to promote cartilage architecture. This proposal introduces a novel live-cell imaging method that allows quantitative analysis of column formation to test the innovative hypothesis that antagonism between Pthrp and Wnt5a signaling regulates myosin II motor protein activity at the cell-cell and cell-matrix interfaces to produce the anisotropic forces required to rearrange daughter chondrocytes into a column. This hypothesis is based in part on the observations of phospho-myosin light chain at the cell-matrix interface during column formation and the relocalization of this activity to the cell-cell interface in Wnt5a mutants and following activation of Pthrp signaling in chondroctyes. The proposed experiments utilize the live- cell imaging system in conjunction with immunofluorescence, electron microscopy, and biochemical analyses to: (1) defining the roles of cell-cell and cell-matrix adhesion in column formation, (2) determining the pathway through which Wnt5a regulates myosin activity in chondrocytes, and (3) testing if Pthrp signaling alters the balance between Rho and Rac GTPase activity at the cell-matrix and cell-cell interfaces, respectively. Results of these studies will advance the development of novel therapeutics, including growth plate cartilage engineering, to treat chondrodysplasias by providing crucial information that provide mechanistic links between known regulators of chondrocyte maturation and cell mechanics that are crucial for generating cartilage tissue architecture and growth.